Western blot

2.1.5.1 Aflatoxins as a health threat to humans and animals

AFs are a group of difuranocoumarin derivatives consisting of 5 heterocycles that occur in several chemical forms. The four major AFs are AFB₁, AFB₂, AFG₁ and AFG₂ (Figure 2.3), and they are named based on their fluorescence under UV light (B for blue and G for green) and relative chromatographic mobility during thin-layer chromatography.

AFB₁ is considered the most potent natural carcinogen known and is usually the major AF produced by aflatoxigenic strains. It is therefore the best studied. Numerous other AFs have been described, especially as mammalian biotransformation products of the major metabolites (Bennett & Klich, 2003). One such example is AFM1, the predominant metabolite of AFB1 in milk from lactating humans and animals that consume AFB1 -contaminated food or feed.

Figure 2.3 Chemical structure of the four major AFs (from: Reiter et al., 2009).

AFB1 is metabolised by the liver through the cytochrome P450 enzyme system to the major carcinogenic metabolite AFB1-8,9-epoxide (AFBO), or to less mutagenic forms such as AFM1, AFQ1, or AFP1 (Shimada & Guengerich, 1989; Crespi et al., 1991). There are several pathways that AFBO can take, resulting in cancer, toxicity, and AFBO excretion.

The exo-form of AFBO readily binds to cellular macromolecules including genetic material (proteins and DNA), to form adducts. It is the formation of DNA-adducts that leads to gene mutations and cancer.

AFs are associated with both toxicity and carcinogenicity in human and animal populations. The diseases caused by AF consumption are called aflatoxicoses. Acute aflatoxicosis occurs when moderate to high levels of AFs are consumed. Acute episodes of disease symptoms may include haemorrhage, acute liver damage, oedema, alteration in digestion, absorption and/or metabolism of nutrients, and may result in death (Varga et al., 2009). Chronic aflatoxicosis results in cancer, immune suppression, and other “slow”

pathological conditions.

There are substantial differences in species susceptibility. Moreover, within a given species, the magnitude of the response is influenced by age, sex, weight, diet, exposure to infectious agents, and the presence of other mycotoxins and pharmacologically active substances. LD50 for AFB₁ ranges from 0.5 mg/kg for the adult dog to 10.2 mg/kg for the hamster (Moss, 1996). For humans, LD50 probably falls in the middle of the range (Moss, 1998). Because of the differences in AF susceptibility in test animals, it has been difficult to extrapolate the possible effects of AFs to humans, but according to the International Agency for Research on Cancer (IARC), there is sufficient evidence for carcinogenicity of naturally occurring mixtures of AFs, mixtures of AFB1, AFG1 and AFM1, and of AFB1

alone, limited evidence for AFB2 and inadequate evidence for AFG2 and AFM1 (IARC, 2002). Exposure to AFs in the diet is considered an important risk factor for the development of primary hepatocellular carcinoma, particularly in individuals already exposed to other liver pathologies such as hepatitis B (Henry et al., 2002). Several studies have linked liver cancer incidence to estimated AF consumption in the diet (Li et al., 2001). The results of these studies have not been entirely consistent, and quantification of lifetime individual exposure to AF is extremely difficult. The incidence of liver cancer varies widely from country to country, but it is one of the most common cancers in China,

the Philippines, Thailand and many African countries (Bennett & Klich, 2003), where contaminated maize and rice are the major dietary constituents.

Also, acute toxicity of AFs in humans has been observed, even if rarely. Acute aflatoxicosis epidemics occurred in India in 1974, due to the consumption of maize heavily contaminated with AF (Krishnamachari et al., 1975). More than 100 people died. Also, three cases of acute aflatoxicosis occurred in Kenya in 1981 (Ngindu et al., 1982), in 2004 and in 2005, causing more than 150 deaths (CDC, 2004; Azziz-Baumgartner et al., 2005;

Lewis et al., 2005; Probst et al., 2007).

2.1.5.2 Risk assessment and Regulatory issues

AFs have been found to contaminate many crops frequently at nanogram levels, although occasionally they can be found at levels of tens to hundreds of ng/g. Commodities with a high risk of AF contamination include peanuts, corn, cottonseed, Brazil nuts, pistachios, spices, figs and copra. Commodities with an intermediate risk of AF contamination include almonds, pecans, and raisins. Walnuts, soybeans, beans, pulses, cassava, grain sorghum, millet, wheat, oats, barley, and rice seem to be less susceptible to AF contamination (CAST, 2003).

Because controlling the occurrence of mycotoxins in finished products is practically impossible, regulatory bodies are continuously assessing the levels of acceptable exposure to humans by using a risk assessment process to establish tolerable daily intakes of selected mycotoxins. Monitoring programs assessing the occurrence of mycotoxins along with available toxicological data are used to make an assessment of exposure-risk to humans or animals. The result is the establishment of regulatory levels for selected mycotoxins where sufficient information has been obtained.

Risk assessment is based on the hazard or toxicity of a mycotoxin and the expected degree of exposure of individuals or populations. The hazard of mycotoxins to individuals is probably more or less the same all over the world, except for those populations, e.g.

from Shanghai, Thailand, China, Gambia, Taiwan, with high levels of hepatitis B infection, for whom AF potency is significantly enhanced (Henry et al., 2002; CAST, 2003). On the other hand, exposure is not the same worldwide, because of different levels of contamination as well as dietary habits in the various parts of the world. AFs prevail in less

developed tropical and subtropical countries where climate and storage conditions are favourable to fungal growth and toxin production. Furthermore, populations from those countries rely extensively on some of those crops which have been found more susceptible to AF, mostly grains.

Worldwide regulations exist for mycotoxins and generally are based on toxicological data, occurrence and distribution, and epidemiological data. In Europe, current regulations are based mostly on scientific opinions of authoritative bodies, as the Joint Expert Committee on Food Additives of the United Nations (JECFA - a scientific advisory body of the World Health Organization (WHO) and the Food and Agriculture Organization (FAO)) and the European Food Safety Authority (EFSA). The EFSA is an independent body of the European Commission (EC), established in 2002, and charged, among other tasks, with the development of risk assessments on issues of concern in the food and feed supply. EFSA publishes its risk assessments in the form of scientific opinions which form the main scientific basis for the preparation of EU regulations. Another important EU activity is SCOOP (Scientific Cooperation on Questions relating to Food), funded by the European Commission, and targeted to make the best estimates of intake of contaminants by EU inhabitants. The objectives of SCOOP activity is to provide the scientific basis for evaluation and management of risk to public health arising from dietary exposure to mycotoxins, taking into account recently available data on occurrence and consumption.

Special emphasis is placed on evaluation of dietary intake of mycotoxins in each of the EU member states and in high-risk sub-groups of the population.

For the mycotoxins currently considered most significant, JECFA has evaluated their hazard in several sessions (see review by van Egmond et al., 2007). In 2001, a JECFA session was devoted to mycotoxins. Reports resulting from this session provided detailed insight into the process of risk assessment of mycotoxins (FAO, 2001, 2002). The reports addressed several concerns about the mycotoxins considered - their properties and metabolism, toxicological studies, and final risk evaluation.

In the early days of mycotoxin regulations, control measures focused mainly on AFs.

They were established by industrialised countries, and limits often had an advisory or guideline character. Over the years, the number of countries with known specific mycotoxin regulations has increased from 33 in 1981 (Schuller et al., 1983) to 100 in 2003

(FAO, 2004), with specific limits being established for many food and feed commodities and products for 13 different mycotoxins or groups of mycotoxins.

Until the late 1990’s, setting of mycotoxin regulations was mostly a national concern. As a consequence, tolerated levels of mycotoxins varied widely between countries. The Task Force Report of the Council for Agricultural Science and Technology (CAST), USA, collated information on almost 80 countries all over the world and, in 1997, for the specific case of AFs, tolerated levels varied from zero (undetectable) to 1000 µ g/kg (CAST, 2003). Preferably, regulations should be harmonised with those in other countries with which trade contacts exist. Unnecessarily strict regulative actions make it difficult for importing countries to obtain supplies of essential commodities such as food grains and animal feedstuffs. Also, exporting countries may have difficulty finding markets for their products. For example, stringent regulations for AFs in the EU (EC, 2006) make it difficult for some countries to export food commodities and feed for their European trading partners. As a consequence, several economic communities, e.g. EU, Mercado Cómun del Sur (MERCOSUR), Australia and New Zealand, have been developing efforts during the last decade in order to harmonise their mycotoxin regulations, thus overruling existing national regulations.

In an attempt for harmonisation, EFSA has recently (March 2007) published an opinion on the potential increase in the risk to consumer health of a possible increase in current maximum levels for AFs in almonds, hazelnuts, pistachios, and derived products (http://www.efsa.europa.eu, accessed 15.07.2010). The panel concluded that changing the maximum levels for total AFs in almonds, hazelnuts, and pistachios from 4 to 8 or 10 µg/kg would have minor effects on estimates of dietary exposure and cancer risks. As a consequence, EC recently adopted legislation changing their AFs regulatory limits (EC, 2010a) and sampling plans (EC, 2010b) for tree nuts to more closely conform to that developed by the Codex Committee on Contaminants in Foods (CCCF) and adopted by the Codex Alimentarius Commission (CAC) in July 2008 (CCCF, 2008). The Codex AF sampling plan for tree nuts (almonds, pistachios, and hazelnuts) requires that two 10 kg samples both test less than 10 µg/kg for total AFs (AFT) to accept the lot. The EU adopted the Codex plan, but added an AFB1 limit of 8 µg/kg. As a result, an almond lot requires two 10 kg samples to each test less than both limits (8 µg/kg AFB1 and 10 µg/kg AFT) for

the lot to be accepted into the food chain. This revision still does not harmonise with USA regulations, which determine maximum levels for total AFs of 20 µ g/kg.

EU food and feed imports are informed in part through the EU’s Rapid Alert System for Food and Feed (RASFF). The RASFF is a tool used to exchange information on potential risks entering the food and feed system at any point in the EU, so that all EU member states may be alerted to take the appropriate measures to assure food and feed safety (Wu, 2008). In 2009, RASFF reported a total of 669 alerts or notifications for mycotoxins, of which 95% were for AFs, mostly from nuts, nut products and seeds (638, 81%) (EC, 2010c). A significant part of these notifications (42%) were for peanuts from Argentina, China, USA, Brazil, Egypt and South Africa. Pistachio nuts from Iran, Turkey and USA originated 136 notifications (21%), 63 notifications (9.9%) on hazelnuts nearly all from Turkey, 55 notifications (8.6%) on almonds mainly from USA and a few from Australia, and 7 notifications (1%) on Brazil nuts from Brazil and Bolivia. The remaining notifications were on figs (10%), spices (3.6%), cereals (2%) and feed (1.4%).

Although this is circumstantial evidence, it reflects market realities and conforms to the position of the industry groups, in which peanut, almond and pistachio producers are greatly affected by the economic impact of AF contamination, whereas others (e.g. walnut producers) are primarily concerned with spoilage microorganisms such as Rhizopus, Penicillium and A. niger.